(P-BC-74) Screening Platelet Donors for HPA-1a Antigen by Flow Cytometry

Background/Case Studies: Human platelet antigens HPA-1a (PLA1) and HPA-1b (PLA2) are a diallelic antigen system on the CD61 antigen, platelet glycoprotein GPIIIa. Anti-HPA-1a antibodies can cause platelet transfusion refractoriness, posttransfusion purpura, and fetal/neonatal alloimmune thrombocytopenia. Since PLA1 is a high-frequency antigen, obtaining PLA1 negative platelets is often challenging. Identifying these rare donors can be prohibitively costly with genetic methodologies. Flow cytometry offers a cost-effective screening method by using a PLA1-specific anti-CD61-FITC antibody.

Study

Design/Methods: Platelet-rich plasma (PRP) was aliquoted from the primary product and stored at room temperature for up to 14 days. Previously tested donors were excluded, with PLA2 and PLA1/A2 specimens serving as negative and positive controls, respectively. Testing was performed using pools of PRP from 4 individual specimens. 200µL from 4 well-mixed specimens were added into a new tube and gently vortexed. 5µL of the Beckman Coulter SZ21 clone anti-CD61-FITC (IM1758U) was added to a new tube, along with 10µL of pooled PRP and 50µL of PBS. The tube was vortexed and incubated in the dark at room temperature for 10 minutes. 500µL of PBS was added to the tube. The tube was then vortexed and analyzed on a Beckman Coulter Navios EX. Smaller platelets were excluded from analysis via a “Platelet” gate on a forward scatter v. side scatter scatterplot (Figure 1). 10,000 large platelet events were analyzed for PLA1 expression, gating on the negative region. Any specimen pools with ≥2% PLA1 negative events were reflexed for testing the individual specimens. Individual specimens with ≥95% negative events were considered PLA1 negative. Peripheral blood specimens on subsequent donations from PLA1 negative donors were confirmed genetically by a reference molecular laboratory using PLA1/2 Single-nucleotide polymorphism (SNP) probes.

Results/Findings: From 10/2023 to 04/2024, we tested 3024 plateletpheresis donors and identified 56 PLA1 negative donors (1.85% frequency). Genetic testing of the 10 most frequent donors confirmed 9 as HPA-1bb and 1 as HPA-1ab (90% concordance). The discrepant donor consistently tested HPA-1a-negative by flow cytometry despite carrying the HPA-1a allele, suggesting a possible mutation affecting epitope expression not detected by the SNP-based test.

Conclusions: Flow cytometry screening for PLA1 expression proved to be quick, affordable, and reliable with 90% accuracy compared to genetic testing. This method allowed efficient screening at approximately one-tenth the cost of molecular methods, enhancing our ability to maintain HPA-1a-negative platelet inventory. Blood centers with flow cytometers may find this approach cost-effective for identifying rare donors lacking this high-frequency antigen.